ProjectSummary/Abstract Necrotizingenterocolitis(NEC)istheleadingcauseofdeathfromgastrointestinaldiseaseinprematureinfants andischaracterizedbyanuncontrolledinflammatoryresponse.Thelackofunderstandingofthemechanisms thatregulatetheviciousinflammatorycascade,theinabilitytodeterminewhichinfantsaresusceptibletoNEC andalackoftherapeutictargetsallcontributetothepersistentlyhighmortalityrate.Thegoalsofthe proposedresearcharetodeterminethemechanismsthatregulatethepathologicimmuneresponse duringNECandusethisknowledgetodesignnovelepithelial-orimmune-specificstrategiesforthis devastatingdisease.Ourlaboratoryhasrecentlydiscoveredthattheinterleukin-22(IL-22)signalingpathway playsacriticalroleinattenuatingtheinflammatoryresponseduringNEC,asmicelackingthereceptorforIL-22 (IL-22Ra1)ontheintestinalepitheliumdemonstrateacceleratedmortalityinaneonatalmousemodelofNEC- likeintestinalinjury.WedemonstratethatmicesubjectedtoexperimentalNECdevelopgrossevidenceof smallintestinalischemiaandnecrosis,thatcanbecompletelyrescuedinmicewithintactIL-22signalingby administeringrecombinantIL-22.Inseekingtodeterminethemechanismsmediatingthisprotection,wehave demonstratedthattreatmentwithrecombinantIL-22decreasesthepro-inflammatoryTh17lymphocytic infiltrate,whichwehaveshowncontributestoNECpathogenesis.Furthermore,wealsodeterminedthatIL-22 signalingisimportantintheregulationofintestinalstemcelldifferentiation,asmicedeficientinIL-22Ra1inthe intestinedemonstrateanabnormallyprofoundphenotypecharacterizedbydecreasednumbersofsecretory cellsintheintestineaswellasdecreasedexpressionofcriticalgenesinvolvedinintestinalstemcell developmentandhostdefense.Basedonthesefindings,wehypothesizethatIL-22signalingthroughthe receptorIL-22Ra1attenuatesNECby1)enhancingintestinalstemcellfunction,2)increasinggobletcell differentiationand3)shiftingtheimmunecellrepertoiretowardsananti-inflammatoryphenotype.Wewill completeouraimsofthisprojectbybringingtogetheramulti-disciplinaryteamwithexpertiseinepithelial biology,mucosalimmunology,masscytometry,next-generationsequencing,high-resolutionconfocaland intravitalmicroscopyaswellasmicrofluidicsandengineeringforthederivationofinvitromodelsofhuman intestinalfunctionusinggut-on-a-chipdevices.Thesestudieswillmakeasignificantconceptualadvancein understandingthesignalingpathwaysinvolvedinattenuatingNEC,explainingtheuniquesusceptibilityofthe prematureinfanttoNECbasedonadefectinIL-22signaling,andwewillevaluateanoveltherapeuticstrategy forNECbyintroducingtheanti-inflammatorycytokineIL-22intheintestinalmilieu.